There have been known saccharides composed of glucose molecules as constituent saccharides, for example, partial starch hydrolysates, produced from starches as materials, including amylose, amylodextrin, maltodextrin, maltooligosaccharide, and isomaltooligosaccharide. Also, these saccharides are known to have usually reducing and non-reducing groups at. their molecular ends and exhibit reducibility. In general, partial starch hydrolysates can be expressed with an index of dextrose equivalent (DE), a scale of reducing power based on the dry solid. Those with a relatively high DE value are usually known to have properties of a relatively low molecular weight, relatively low viscosity, strong sweetness and reactivity, easy reactivity with amino group-containing substances such as amino acids and proteins that may induce browning and unpleasant smell, and easily cause deterioration. To improve these defects, there has long been desired a method for lowering or eliminating the reducing power without altering glucose molecules as constituent saccharides of partial starch hydrolysates. For example, as disclosed in Journal of American Chemical Society, Vol. 71, pp. 353–358 (1949), it was reported that a method for forming α-, β-, and γ-cyclodextrins that are composed of 6–8 glucose molecules linked together via the α-1,4 glucosidic linkage by contacting amylases, derived from microorganisms of the species Bacillus macerans, with starches. Nowadays, these cyclodextrins are produced on an industrial scale and used in diversified fields using their inherent properties such as non-reducibility, tasteless, and enclosing ability. As disclosed, for example, in Japanese Patent Kokai Nos. 143,876/95 and 213,283/95 applied for by the same applicant as the present invention, it is known a method for producing trehalose, composed of two glucose molecules linked together via the α,α-linkage, by contacting a non-reducing saccharide-forming enzyme and a trehalose-releasing enzyme with partial starch hydrolysates such as maltooligosaccharide. At present, trehalose has been industrially produced from starches and used in different fields by using its advantageous non-reducibility, mild- and high quality-sweetness. As described above, trehalose having a glucose polymerization degree (DP) of two, and α-, β-, and γ-cyclodextrins having a DP of 6–8 are produced on an industrial scale and used in view of their advantageous properties, however, the types of non- or low-reducing saccharides are limited, so that more diversified saccharides other than these saccharides are greatly required.
Recently, a new type of cyclotetrasaccharide, composed of glucose units, was reported. European Journal of Biochemistry, Vol. 226, pp. 641–648 (1994) shows that a cyclic tetrasaccharide having the structure of cyclo{→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→} (may be called “cyclotetrasaccharide” throughout the specification) is formed by contacting a hydrolyzing enzyme, alternanase, with alternan linked with glucose molecules via the alternating α-1,3 and α-1,6 bonds, followed by crystallization in the presence of methanol as an organic solvent.
Cyclotetrasaccharide or a non-reducing saccharide having a cyclic structure, exhibits an inclusion ability to stabilize volatile organic compounds, and does not cause an amino carbonyl reaction, and therefore it is expected to be used and processed with lesser fear of browning and deterioration.
However, the material alternan and alternanase, which are needed for producing the saccharide, are not easily obtainable, and the microorganisms for their production are not easily available.
Under such conditions, the present inventors succeeded in producing cyclotetrasaccharide by contacting, as a material, a saccharide having the α-1,6 glucosidic linkage as a linkage of non-reducing end and having a glucose polymerization degree of at least three (may be called “α-isomaltosylglucosaccharide” throughout the specification) with an α-isomaltosyl-transferring enzyme which specifically hydrolyzes the α-isomaltosyl moiety and the resting glucosylsaccharide moiety and then transfers the α-isomaltosyl moiety to its acceptor to form cyclotetrasaccharide, as disclosed in Japanese Patent Application Nos. 149,484/2000 and 229,557/2000. The α-isomaltosyl-transferring enzyme is an enzyme which forms cyclotetrasaccharide from α-isomaltoglucosaccharide by α-isomaltosyl-transferring reaction and has the following physicochemical properties:
(1) Action                Forming cyclotetrasaccharide having the structure of cyclo{→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→} from a saccharide having a glucose polymerization degree of at least three and having both the α-1,6 glucosidic linkage as a linkage at the non-reducing end and the α-1,4 glucosidic linkage other than the above linkage;        
(2) Molecular Weight                Having a molecular weight of about 82,000 to about 136,000 daltons when determined on sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE);        
(3) Isoelectric Point (pI)                Having a pI of about 3.7 to about 8.3 when determined on isoelectrophoresis using ampholine;        
(4) Optimum Temperature                Having an optimum temperature of about 45° C. to about 50° C. when incubated at a pH of 6.0 for 30 min;        
(5) Optimum pH                Having an optimum pH of about 5.5 to about 6.5 when incubated at 35° C. for 30 min;        
(6) Thermal Stability                Having a thermostable range at temperatures of about 45° C. or lower when incubated at a pH of 6.0 for 60 min; and        
(7) pH Stability                Having a stable pH range at about 3.6 to about 10.0 when incubated at 4° C. for 24 hours.        
Referring to the material saccharides for cyclotetrasaccharide, it should desirably be produced from the abundant and low-cost starches, however, since α-isomaltosyl-transferring enzyme does not directly act on starches, the following procedure is actually employed: Starches are first converted into such an α-isomaltosylglucosaccharide having the above specified structure, for example, relatively-low molecular weight isomaltooligosaccharides such as panose and isomaltosylmaltose, and then subjected to the action of α-isomaltosyl-transferring enzyme to form cyclotetrasaccharide.
It was found that, when used panose as a material for cyclotetrasaccharide, the yield of the saccharide from the material is about 44% to the material, based on the weight of the dry solid (d.s.b.). Similarly, in the case of using isomaltosylmaltose as a material, the yield of cyclotetrasaccharide is about 31%, d.s.b., while in the case of using starches as a material, they should be contacted with enzymes such as α-amylase, starch debranching enzyme, β-amylase, and α-glucosidase to form relatively-low molecular weight isomaltooligosaccharides including panose, and the yield of cyclotetrasaccharide is quite as low as about 15%, d.s.b.
Although the actual production of cyclotetrasaccharide is feasible from starches even with such a low yield, the production cost may be increased. Under these circumstances, it is desired to establish a novel method for producing cyclotetrasaccharide in a relatively high yield using easily available materials such as starches.